The long-term goals of Project 2 are to understand the effects of diet and genes on variation in risk for cardiovascular disease (CVD). Our research to date has focused on localizing genes underlying variation in lipids, lipoproteins, oxidative stress and inflammation, which are among the strongest, most consistent predictors of CVD in the epidemiological literature. However, many of these detected genes act in concert to influence complex biological systems. Our central hypothesis is that there are pleiotropic networks of coordinately-regulated genes and clinical risk factor phenotypes that influence susceptibility to CVD. We propose to apply a systems biology-based approach to address this hypothesis. In Aim 1, we will construct pleiotropic networks of genes underlying correlated responses of lipoproteins and related CVD risk factors to dietary cholesterol, fat, and anti-oxidant supplementation. Utilizing data from completed dietary challenge experiments and from whole-genome transcriptional profiles for 500 baboons, we will construct networks of clinical phenotypes and expression profiles, and compare these networks to evaluate the relationships between expression and risk factor variation. In a newly added sub-aim, we will validate these results by analyzing transcriptional profile data and phenotype measures already in hand from a large-scale study of the genetics of CVD risk factors in extended human families. In Aim 2, we will construct pleiotropic networks of genes underlying co-variation in functional markers of the vascular endothelium. Phenotypes include endothelial progenitor cell numbers, ex vivo primary endothelial cell (EC) properties plus in vivo functional indicators of the vascular endothelium, including vascular reactivity and circulating levels of VCAM, ICAM, and vWF. We also will determine transcriptional profiles from primary ECs in order to directly compare networks of expression and risk factor phenotypes. In Aim 3 we will evaluate contributions of risk factors and biological network components identified in Aims 1 and 2 to variation in extent of lesions following a chronic, 2-year diet challenge. Data for this aim include measures related to lipoprotein metabolism and oxidative damage, assessments of endothelial function, and EC transcriptional profiles. Additionally, as part of this aim, we also will assess the effects of acute exposure (7-weeks) to the atherogenic diet on EC gene expression and, if such effects are detected, we will test for their relationships to later lesion formation and to gene expression in another critical tissue or CVD, the liver. Completion of these aims will provide valuable insights into the interrelationships between known and novel CVD risk factors in general; identify networks of genes whose expression is affected by dietary fat; validate these networks and their components in humans; and examine the relationships between dietary fat, EC gene expression, and arterial lesion formation.